Coupling of ion pair ultrasound assisted-cloud point extraction to microvolume UV-Vis spectrophotometry for speciation analysis of ionic NO2-,NO3- and total NO2-/NO3- without and with reduction in the selected beverage and food matrices

This study proposes a simple, low-cost, easy to use, fast, accurate and reliable ultrasound assisted-cloud point extraction (UA-CPE) method for the enrichment and speciation analysis of low levels of ionic NO₂ ⁻, NO₃ ⁻ and total NO₂ ⁻/NO₃ ⁻ without and with reduction by micro-volume UV-vis spectrophotometry. The method relies on the reaction of NO₂ ⁻ with excess iodide at pH 5.5 to form triiodide, and then the extraction of the oxidation product formed by charge transfer into micellar phase of Triton X-114 using cationic redox-sensitive phenazine dye, neutral red (NRH⁺) as ion-pairing in absence and presence of 1.5 mL of 1.0 × 10⁻³ mol L⁻¹ Br⁻ ions. The main variables affecting extraction efficiency were studied and optimised in detail. Under the optimal conditions, the calibration curves in solvent were linear over the ranges of 5–100 and 100–1280 µg L⁻¹ with a detection limit of 1.32 µg L⁻¹ while the matrix-matched calibration curves prepared from sample extracts were linear over the ranges of 5–125 and 125–1350 µg L⁻¹ with a detection limit ranging from 1.15 to 1.50 µg L⁻¹ for sample matrices. From pre-concentration of 25-mL sample, pre-concentration and sensitivity enhancement folds of 62.5 and 76.5 were obtained. The accuracy/precision studies after spiking were performed, and observed to be in range of 96.5–99.6% and 1.9–5.7% (15, 75 and 250 µg L⁻¹, n: 5). The speciation analysis of ionic NO₂ ⁻, NO₃ ⁻ and total NO₂ ⁻/NO₃ ⁻ was performed from artificially prepared binary mixtures after reduction with a mixture of Zn and NaCl in acidic media. The free NO₃ ⁻ contents of samples were calculated from difference between free NO₂ ⁻ and total NO₂ ⁻/NO₃ ⁻ contents. After validation by analysis of a certified sample, the method was successfully applied to the speciation analysis of the beverage, cured meat, vegetable and milk samples. After pre-treatment with two sample preparation steps, it was observed that the results obtained were also compatible with each other, statistically validating the method.